A refined understanding of virus/host relationships that promote transmission and those that contribute to the rate of disease progression following infection is critical for the design of effective vaccines against HIV-1. One parameter that is increasingly recognized as being critical and understudied is the role of viral env glycosylation not only in preferential transmission but also in the ability of the virus to select receptors for entry and for its susceptibility to neutralizing antibodies. In addition, data is now accumulating o show that a variety of cell surface molecules that serve as ligands/receptors for the glycosylated env proteins and those that are influenced by glycosylated versus de-glycosylated viruses, could contribute to the quality of immune response the virus engenders. These issues can only be objectively addressed using appropriate nonhuman primate models such as macaques infected with SHIVs that contain the env from primary HIV-1 isolates, particularly from transmitted/founder viruses to study the roles of env glycosylation in preferential transmission. Therefore, we plan to explore the role of env glycans using sets of HIV molecular clones obtained from transmitted/founder (T/F) viruses from a Zambian cohort-where clade C viruses predominate. We have generated several unique tools/reagents and assembled a highly talented team of Investigators and a series of systematic logically designed studies are outlined which will first focus on the composition and arrangement of glycans in paired clones from T/F circulating HIV strains including N-and O-glycans as outlined in aim 1. Based on glycan profiles, we proposed to select a) a highly glycosylated, b) low level of env glycosylated viruses from transmitter and c) as control, a founder virus to prepare corresponding replication competent Simian Human Immunodeficiency viruses (SHIV) for further advanced studies, which include detailed in vitro characterization of replication kinetics in primary cells and cell lines, glycan content, particle envelope glycoprotein content, dendritic cell (DC) capture, DC-T cell trans-infection, sensitivity to IFN-?, anti-?47 and blocking mAbs against select lectin-like molecules (Galectin-9, SIGLECs, MINCLE, and CD200/200R). Then we will select the viruses to test in vivo for transmission efficiency studies in rhesus macaques based primarily on comparable in vitro replication but differing glycan content (aim 2). Under specific aim 3, we will define whethe it is the signature site(s) of Env glycosylation that facilitates transmission, using isogenic SHIV based on the backbone of the poor transmitter and mutating only those select residues corresponding to the founder virus from aim 2b, using a repeated multiple-low-dose Intra-Vaginal (IVAG) challenge model. Results of these studies will provide a strong foundation for future rational HIV vaccine design.